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Cellular and molecular genetic approaches in Alzheimer's disease
Neurobioiogy of Aging, Vol. 15, Suppl. 2. pp. S135.-S137, 1994 Copyright ~ 1994. Elsevier Science Ltd Printed in the USA. All rights reserved 0197-4580/94 S6,00 + .00
Pergamon 0197-4580(94)00085-9
Cellular and Molecular Genetic Approaches in Alzheimer's Disease S A N G R A M S. SISODIA
Neuropathology Labratory, Department of Pathology, The Johns Hopkins University School of Medicine, 558 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196 ALZHEIMER'S disease (AD) is the most common form of dementia and is, therefore, a major cause of disability and death in the elderly. Neurobiological, cellular, and molecular biological, and genetic studies have clarified several aspects of AD, including the vulnerability of specific neural systems, abnormalities in cytoskeletal proteins, metabolism of the amyloid precursor protein (APP) and the [3-amyloid protein (A~), and the genetic linkage of specific mutations and chromosomal loci to the disease. More recently, several investigations have attempted to develop animal models that recapitulate features of the human disease. This document is intended to provide both an overview of the recent progress in some of these areas of research and a discussion of approaches to clarify underlying pathogenetic mechanisms of AD. Over the past few years, research has clarified several aspects of APP processing and AI3 production in cultured cells. Despite these insights, several additional issues require our undivided attention, including the identification of specific proteases involved in APP processing and their intracellular site(s) of action and characterization of APP trafficking pathways in neuronal and nonneuronal cells. For example, it is now clear that a fraction of APP are proteolytically cleaved within the AI3 sequence to generate secreted soluble APP derivatives (1,6,23,31,34) and that a large fraction of APP is metabolized by endosomal/lysosomal pathways (5,8,9). Recent studies have indicated that AI3 is normally secreted from cultured cells and is detectable in human cerebrospinal fluid (10,20,22). Given these advances, it has been particularly disappointing that information regarding the proteases involved in cleavage within the AI3 sequence or at the termini of AI3 (13- and ~/-secretases, respectively) are unavailable. Clearly, these proteases are potential therapeutic targets, and modulation of their activities may have the potential to lessen the amyloid burden in affected individuals. In this vein, recent studies have described several missense mutations in APP that cosegregate with early onset familial AD (3,7,11,14,15) and cerebral hemorrhage (13,30) inherited in an autosomal dominant mode. Studies have clearly demonstrated that the metabolism of APP harboring some of these mutations is altered relative to wild-type APP and results in increased AI3 production (2,4) or secretion of C-terminally extended AI3 (29). With the realization that APP processing is modified by the presence of these mutations, biochemical and pharmacological strategies aimed at identifying target proteases now seem obvious. Alternatively, as biochemical studies on protease activities in lysed cells are inherently difficult, it may be wise to attempt to characterize APP-specific proteases using well-established genetic assays (i.e., mutagenesis and complementation) in yeast. Finally, our knowledge of intracellular trafficking of APP and the cellular site(s) of the processing events in cultured cells, in primary culS135
tures, and in vivo are not completely defined, and clarification of these issues using sophisticated cell biological approaches are desirable. Despite the wealth of information pertaining to APP metabolism, remarkably little is known about the physiological function(s) of APP in the nervous system and in peripheral tissues. For example, APP has been suggested to play a role as a receptor involved in cell-cell communication/synaptic interactions (19,21). It would be highly informative to address issues pertaining to the expression of APP during vertebrate development, particularly in species that have proven amenable to molecular and genetic manipulation (e.g., Xenopus, zebrafish, and mouse). More information is needed concerning the cellular and subcelluar distribution of APP in mature organisms and the modulation of its expression following nerve or excitotoxic lesions and iscbemic injury. In parallel, recent studies have described the expression of APP homologues, termed amyloid precursor-like protein (APLP)I and APLP2, in vertebrates (25,26,32,33), and examination of the metabolism and developmental expression of these polypeptides is warranted. It is equally important to critically reevalute several earlier studies of APP localization and metabolism, given recent concerns regarding antibody cross reactivity between homologues
(25). Recent studies have identified two other genetic loci important for the pathogenesis of AD. One locus on the distal part of chromosome 14 is linked to the majority of early onset disease (18). A susceptibility locus on chromosome 19 associated with late-onset kindreds and sporadic AD has also been described (17). In these late-onset kindreds, a significant association exists between the presence of the apolipoprotein E type-4 allele and the presence of AD. Obviously, considerable financial resources and effort need to be expended towards identification of the gene on chromosome 14. The role of apoE in the pathogenesis of late-onset AD is presently uncertain, although several in vitro studies have implicated varying affinity of apoE for AI3 (27) and/or a microtubuleassociated protein, tau (28). Several of these hypotheses can be immediately tested in cotransfection assays. Parallel studies of APP processing in these systems might provide valuable insights into potential overlaps in the biology of APP and apoE. A final issue that requires considerable emphasis relates to the development of transgenic animal models of AD (16). At present, the nonhuman primate is the most useful animal model for examining the role of aging in the behavioral and histopathological abnormalities that resemble those occuring AD. However, these animals are not particularly valuable for biochemical and molecular studies or testing of therapeutic agents because these animals are extremely scarce, the maintenance costs are enormous, and
S 136
SISODIA
they exhibit highly variable pathology and behaviors. On the other hand, transgenic mice might provide a powerful model that recapitulates the pathologic features of AD. Although recent studies have demonstrated that human APP can be expressed in transgenic mice (12,24), these animals do not, as yet, exhibit any pathologic features of AD. Future strategies should include generation of mice that express APP variants described in individuals with early onset familial AD. In addition, transgenic studies involving the introduction of the gene(s) on chromosome 14 and various apoE alleles should be performed. Additional strategies involving crossbreeding of different transgenic lines or breeding transgenic lines to mice containing mutationally inactivated endogenous sequences may accelerate the temporal progression and/or exacerbate the pathology of disease. In most cases, it is clear that this field will derive most benefit by inviting established cellular and molecular biologists, developmental biologists, and geneticists into the community to investigate different issues relevent to the pathogenesis and/or etiology of AD. I strongly suggest that the NIA promote interdisciplinary minisymposia that include both investigators in AD and experts in specific areas of molecular biology/biochemistry and genetics. Examples of such a forum were the NIA-sponsored meeting on Pro-
teases/Protease Inhibitors in AD (December, 1991) and the Symposium on AD and Neuronal Cell Biology (sponsored by the Adler Foundation, February, 1994). To encourage participants from outside the field to actively participate in AD research, mechanisms should be provided to enable those individuals to receive financial support. Issuing RFA directed towards issues pertinent to this disease (i.e.. trafficking and metabolism of membrane glycoproteins, lipoprotein metabolism, biology of neuronal/glial/ endothelial cells, proteases and protease inhibitors, and transgenic models) would be the most direct and satisfying mechanism to elicit the enthusiasm of scientists in the basic science arena. It is imperative that we focus on fundamental aspects of the genetics and biochemistry of AD; insights gained from these basic approaches will, indoubtedly, impact on our understanding of underlying pathogenetic mechanisms and the development and testing of new therapies for this devastating human illness.
ACKNOWLEDGEMENTS This work was supported by grants from the U.S. Public Health Service (NIH NS 20471 and AG 05146) and the Adler Foundation.
REFERENCES l. Anderson, J. P.; Esch, F. S.; Keim, P. S.; Sambamurti, K.; Lieberburg, I.; Robakis, N. K. Exact cleavage site of Alzheimer amyloid precursor in neuronal PC-12 cells. Neurosci. Len. 128:126-128; 1991. 2. Cai, X.-D.; Golde, T. E.; Younkin, S. G. Release of excess amyloid 13 protein from a mutant amyloid 13 protein precursor. Science 259: 514-516; 1993. 3. Chartier-Harlin, M.-C.; Crawford, F.; Houlden, H.; Warren, A.; Hughes, D.; Fidani, L.; Goate, A.; Rossor, M.; Roques, P.; Hardy, J.; Mullan, M. Early onset Alzheimer's disease caused by mutations at codon 717 of the 13-amyloid precursor protein gene. Nature 353: 844-846; 1991. 4. Citron, M.; Oltersdorf, T.; Haass, C.; McConlogue, L., Hung, A. Y.; Seubert, P.; Vigo-Pelfrey, C.; Lieberburg, I.; Selkoe, D. J. Mutation of the 13-amyloid precursor protein in familial Alzheimer's disease increases 13-protein production. Nature 360:672-674; 1992. 5. Cole, G. M.; Huynh, T. V.; Saitoh, T. Evidence for lysosomal processing of amyloid 13-protein precursor in cultured cells. Neurochem. Res. 14:933-939; 1989. 6. Esch, F. S.; Keim, P. S.; Beanie, E. C.; Blacher, R. W.; Culwell, A. R.; Oltersdorf, T.; McClure, D.; Ward, P. J. Cleavage of amyloid 13peptide during constitutive processing of its precursor. Science 248: 1122-1124; 1990. 7. Goate, A.; Chartier-Harlin, M.-C.; Mullah, M.; Brown, J.; Crawford, F.; Fidani, L.; Giuffra, L.; Haynes, A.; Irving, N.; James, L.; Mant, R.; Newton, P.; Rooke, K.; Roques, P.; Talbot, C.; Pericak-Vance, M.; Roses, A.; Williamson, R.; Rossor, M.; Owen, M.; Hardy, J. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature 349:704-706; 1991. 8. Golde, T. E.; Estus, S.; Younkin, L. H.; Selkoe, D. J.; Younkin, S. G. Processing of the amyloid protein precursor to potentially amyloidogenic derivatives. Science 255:728-730; 1992. 9. Haass, C.; Koo, E. H.; Mellon, A.; Hung, A. Y.; Selkoe, D. J. Targeting of cell-surface 13-amyloid precursor protein to lysosomes: AIten.tative processing into amyloid-bearing fragments. Nature 357:500503; 1992. 10. Haass, C.; Schlossmacher, M. G.; Hung, A. Y.; Vigo-Pelfrey, C.; Mellon, A.; Ostaszewski, B. L.; Lieberburg, I.; Koo, E. H.; Schenk, D.; Teplow, D. B.; Selkoe, D. J. Amyloid 13-peptide is produced by cultured cells during normal metabolism. Nature 359:322-325; 1992. 11. Hendricks, L.; van Duijn, C. M.; Cras, P.; Cruts, M.; Van Hul, W.; van Harskamp, F.; Warren, A.; Mclnnis, M. G.; Antonarakis, S. E.; Martin, J.-J.; Hofman, A.; Van Broeckhoven, C. Presenile dementia and cerebal haemorrhage linked to a mutation at codon 692 of the 13-amyloid precursor protein gene. Nature Genet. 1:218-221; 1992.
12. Lamb, B. T.; Sisodia, S. S.; Lawler, A. M.; Slum, H. H.; Kin, C. A.; Keams, W. G.; Pearson, P. L.; Price, D. L.; Gearhart, J. D. Introduction and expression of the 400 kilobase precursor amyloid protein gene in transgenic mice. Nature Genet. 5:22-30; 1993. 13. Levy, E.; Carman, M. D.; Fernandez-Madrid, I. J.; Power, M. D.; Lieberburg, I.; van Duinen, S. G.; Bots, G. T. A. M.; Luyendijk, W.; Frangione, B. Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral hemorrage, Dutch type. Science 248:t1241126. 14. Mullan, M.; Crawford, F.; Axelman, K.; Houlden, H.; Lillius, L.; Winblad, B.; Lannfelt, L. A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of 13-amyloid. Nature Genet. 1:345--347; 1992. 15. Naruse, S.; lgarashi, S.; Kobayashi, H.; Aoki, K.; Inuzuka, T.; Kaneko, K.; Shimizu, T.; Iihara, K.; Kojima, T.; Miyatake, T.; Tsuji, S. Mis-sense mutation Val->lle in exon 17 of amyloid precursor protein gene in Japanese familial Alzheimer's disease. Lancet 337:978-979; 1991. 16. Price, D. L.; Sisodia, S. S. Cellular and molecular biology of Alzheimer's disease and animal models. Annu. Rev. Med. 45:435-.--446; 1994. 17. Saunders, A. M.; Strittmatter, W. J.; Schmechel, D.; St.GeorgeHyslop, P. H.; Pericak-Vance, M. A.; Joo, S. H.; Rosi, B. L.; Gusella, J. F.; Crapper-MacLachlan, D. R.; Alberts, M. J.; Hulette, C.; Crain, B.; Goldgaber, D.; Roses, A. D. Association of apolipoprotein E allele ~4 with late-onset familial and sporadic Alzheimer's disease. Neurology 43:1467-1472; 1993. 18. Schellenberg, G. D.; Bird, T. D.; Wijsman, E. M.; On', H. T.; Anderson. L.; Nemens, E.; White, I. A.; Bonnycastle, L.; Weber, J. L.; Alonso, M. E.; Potter, H.; Heston, L. L.; Martin, G. M. Genetic linkage evidence for a familial Alzheimer's disease locus on chomosome 14. Science 258:668--671; 1992. 19. Schubert; D.; Jin, L.-W.; Saltoh, T.; Cole, G. The regulation of amyloid 13 protein precursor secretion and its modulatory role in cell adhesion. Neuron 3:689-694; 1989. 20. Seubert, P.; Oltersdorf, T.; Lee, M. G.; Barbour, R.; Blomquist, C.; Davis, D. L.; Bryant, K.; Fritz, L. C.; Galasko, D.; Thai, L. J.; Lieberburg. I.; Schenk, D. B. Secretion of 13-amyloid precursor protein cleaved at the amino terminus of the 13-amyloid peptide. Nature 361:260--263; 1993. 21. Shivers, B. D.; Hilbich, C.; Multhaup, G.; Salbaum, M.; Beyreuther. K.; Seeburg. P. H. Alzheimer's disease amyloidogenic glycoprotein: Expression pattern in rat brain suggests a role in cell contact. EMBO J. 7:1365-1370; 1988. 22. Shoji, M.: Golde, T. E.; Ghiso, J.; Cheung, T. T.; Estus, S.; Shaffer,
CELLULAR AND GENETIC APPROACHES IN AD
23. 24. 25.
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L. M., Cai, X.-D.; McKay, D. M.; Tinmer, R.; Frangione, B.; Younkin. S. G. Production of the Alzheimer amyloid 13 protein by normal proteolytic processing. Science 258:126-129; 1992. Sisodia. S. S.; Koo, E. H.; Beyreuther, K.; Unterbeck, A.; Price, D. L. Evidence that 13-amyloid protein in Alzheimer's disease is not derived by normal processing. Science 248:492-495; 1990. Sisodia, S. S.; Price, D. L. Amyloidogenesis in Alzheimer's disease: Basic biology and animal models. Curt. Opin. Neurobiol. 2:648-652; 1992. Slunt, H. H.; Thinakaran, G.; von Koch, C.; Lo, A. C. Y.; Tanzi, R. E.; Sisodia, S. S. Expression of a ubiquitous, cross-reactive homologue of the mouse 13-amyloid precursor protein (APP). J. Biol. Chem. 269:2637-2644; 1994. Sprecher. C. A.; Grant, F. J.; Grimm, G.; O'Hara, P. J.; Norris, F.; Norris, K.; Foster, D. C. Molecular cloning of the cDNA for a human amyloid precursor protein homolog: Evidence for a multigene family. Biochemistry 32:4481---4486; 1993. Strittmatter, W. J.; Saunders, A. M.; Schmechel, D.; Pericak-Vance, M.; Enghild, J.; Salvesen, G. S.; Roses, A. D. Apolipoprotein E: High-avidity binding to 13-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc. Natl. Acad. Sci. USA 90:1977-1981; 1993. Strittmatter, W. J.; Weisgraber, K. H.; Goedert, M.; Saunders, A. M.; Huang, D.; Corder, E. H.; Dong, L.-M.; Jakes, R.; Alberts, M. J.; Gilbert, J. R.; Han, S.-H.; Hulette, C.; Einstein, G.; Schmechel, D. E.; Pericak-Vance, M. A.; Roses, A. D. Hypothesis: Microtubule instability and paired helical filament formation in the
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29.
30.
31. 32.
33.
34.
Alzheimer disease brain are related to apolipoprotein E genotype. Exp. Neurol. 125:163-171; 1994. Suzuki, N.; Cheung, T. T.; Cal, X.-D.; Odaka, A.; Otvos, L., Jr.; Eckman, C.; Golde, T. E.; Younkin, S. G. An increased percentage of long amyloid 13 protein secreted by familial amyloid 13 protein precursor (13APP717) mutants. Science 264:1336.--1340; 1994. Van Broeckhoven, C.; Haan, J4 Bakker, E.; Hardy, J. A.; Van Hul, W.; Wehnert, A.; Vegter-Van der Vlis, M.; Roos, R. A. C. Amyloid 13 protein precursor gene and hereditary cerebral hemorrhage with amyloidosis (Dutch). Science 248:1120-1122; 1990. Wang, R.; Meschia, J. F.; Cotter, R. J.; Sisodia, S. S. Secretion of the 13/A4amyloid precursor protein. Identification of a cleavage site in cultured mammalian cells. J. Biol. Chem. 266:16960--16964; 1991. Wasco, W.; Bupp, K.; Magendantz, M.; Gusella, J. F.; Tanzi, R. E.; Solomon, F. Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid-betaprotein precursor. Proc. Natl. Acad. Sci. USA 89:10758-10762; 1992. Wasco, W.; Gurubhagavatula, S.; Paradis, M. D.; Romano, D. M.; Sisodia, S. S.; Hyman, B. T.; Neve, R. L.; Tanzi, R. E. Isolation and characterization of APLP2 encoding a homologue of the Alzheimer's associated amyloid 13protein precursor. Nature Genet. 5:95-99; 1993. Weidemann, A.; K6nig, G.; Bunke, D.; Fischer, P.; Salbaum, J. M.; Masters, C. L.; Beyreuther, K. Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein. Cell 57:115-126; 1989.
Pergamon 0197-4580(94)00085-9
Cellular and Molecular Genetic Approaches in Alzheimer's Disease S A N G R A M S. SISODIA
Neuropathology Labratory, Department of Pathology, The Johns Hopkins University School of Medicine, 558 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196 ALZHEIMER'S disease (AD) is the most common form of dementia and is, therefore, a major cause of disability and death in the elderly. Neurobiological, cellular, and molecular biological, and genetic studies have clarified several aspects of AD, including the vulnerability of specific neural systems, abnormalities in cytoskeletal proteins, metabolism of the amyloid precursor protein (APP) and the [3-amyloid protein (A~), and the genetic linkage of specific mutations and chromosomal loci to the disease. More recently, several investigations have attempted to develop animal models that recapitulate features of the human disease. This document is intended to provide both an overview of the recent progress in some of these areas of research and a discussion of approaches to clarify underlying pathogenetic mechanisms of AD. Over the past few years, research has clarified several aspects of APP processing and AI3 production in cultured cells. Despite these insights, several additional issues require our undivided attention, including the identification of specific proteases involved in APP processing and their intracellular site(s) of action and characterization of APP trafficking pathways in neuronal and nonneuronal cells. For example, it is now clear that a fraction of APP are proteolytically cleaved within the AI3 sequence to generate secreted soluble APP derivatives (1,6,23,31,34) and that a large fraction of APP is metabolized by endosomal/lysosomal pathways (5,8,9). Recent studies have indicated that AI3 is normally secreted from cultured cells and is detectable in human cerebrospinal fluid (10,20,22). Given these advances, it has been particularly disappointing that information regarding the proteases involved in cleavage within the AI3 sequence or at the termini of AI3 (13- and ~/-secretases, respectively) are unavailable. Clearly, these proteases are potential therapeutic targets, and modulation of their activities may have the potential to lessen the amyloid burden in affected individuals. In this vein, recent studies have described several missense mutations in APP that cosegregate with early onset familial AD (3,7,11,14,15) and cerebral hemorrhage (13,30) inherited in an autosomal dominant mode. Studies have clearly demonstrated that the metabolism of APP harboring some of these mutations is altered relative to wild-type APP and results in increased AI3 production (2,4) or secretion of C-terminally extended AI3 (29). With the realization that APP processing is modified by the presence of these mutations, biochemical and pharmacological strategies aimed at identifying target proteases now seem obvious. Alternatively, as biochemical studies on protease activities in lysed cells are inherently difficult, it may be wise to attempt to characterize APP-specific proteases using well-established genetic assays (i.e., mutagenesis and complementation) in yeast. Finally, our knowledge of intracellular trafficking of APP and the cellular site(s) of the processing events in cultured cells, in primary culS135
tures, and in vivo are not completely defined, and clarification of these issues using sophisticated cell biological approaches are desirable. Despite the wealth of information pertaining to APP metabolism, remarkably little is known about the physiological function(s) of APP in the nervous system and in peripheral tissues. For example, APP has been suggested to play a role as a receptor involved in cell-cell communication/synaptic interactions (19,21). It would be highly informative to address issues pertaining to the expression of APP during vertebrate development, particularly in species that have proven amenable to molecular and genetic manipulation (e.g., Xenopus, zebrafish, and mouse). More information is needed concerning the cellular and subcelluar distribution of APP in mature organisms and the modulation of its expression following nerve or excitotoxic lesions and iscbemic injury. In parallel, recent studies have described the expression of APP homologues, termed amyloid precursor-like protein (APLP)I and APLP2, in vertebrates (25,26,32,33), and examination of the metabolism and developmental expression of these polypeptides is warranted. It is equally important to critically reevalute several earlier studies of APP localization and metabolism, given recent concerns regarding antibody cross reactivity between homologues
(25). Recent studies have identified two other genetic loci important for the pathogenesis of AD. One locus on the distal part of chromosome 14 is linked to the majority of early onset disease (18). A susceptibility locus on chromosome 19 associated with late-onset kindreds and sporadic AD has also been described (17). In these late-onset kindreds, a significant association exists between the presence of the apolipoprotein E type-4 allele and the presence of AD. Obviously, considerable financial resources and effort need to be expended towards identification of the gene on chromosome 14. The role of apoE in the pathogenesis of late-onset AD is presently uncertain, although several in vitro studies have implicated varying affinity of apoE for AI3 (27) and/or a microtubuleassociated protein, tau (28). Several of these hypotheses can be immediately tested in cotransfection assays. Parallel studies of APP processing in these systems might provide valuable insights into potential overlaps in the biology of APP and apoE. A final issue that requires considerable emphasis relates to the development of transgenic animal models of AD (16). At present, the nonhuman primate is the most useful animal model for examining the role of aging in the behavioral and histopathological abnormalities that resemble those occuring AD. However, these animals are not particularly valuable for biochemical and molecular studies or testing of therapeutic agents because these animals are extremely scarce, the maintenance costs are enormous, and
S 136
SISODIA
they exhibit highly variable pathology and behaviors. On the other hand, transgenic mice might provide a powerful model that recapitulates the pathologic features of AD. Although recent studies have demonstrated that human APP can be expressed in transgenic mice (12,24), these animals do not, as yet, exhibit any pathologic features of AD. Future strategies should include generation of mice that express APP variants described in individuals with early onset familial AD. In addition, transgenic studies involving the introduction of the gene(s) on chromosome 14 and various apoE alleles should be performed. Additional strategies involving crossbreeding of different transgenic lines or breeding transgenic lines to mice containing mutationally inactivated endogenous sequences may accelerate the temporal progression and/or exacerbate the pathology of disease. In most cases, it is clear that this field will derive most benefit by inviting established cellular and molecular biologists, developmental biologists, and geneticists into the community to investigate different issues relevent to the pathogenesis and/or etiology of AD. I strongly suggest that the NIA promote interdisciplinary minisymposia that include both investigators in AD and experts in specific areas of molecular biology/biochemistry and genetics. Examples of such a forum were the NIA-sponsored meeting on Pro-
teases/Protease Inhibitors in AD (December, 1991) and the Symposium on AD and Neuronal Cell Biology (sponsored by the Adler Foundation, February, 1994). To encourage participants from outside the field to actively participate in AD research, mechanisms should be provided to enable those individuals to receive financial support. Issuing RFA directed towards issues pertinent to this disease (i.e.. trafficking and metabolism of membrane glycoproteins, lipoprotein metabolism, biology of neuronal/glial/ endothelial cells, proteases and protease inhibitors, and transgenic models) would be the most direct and satisfying mechanism to elicit the enthusiasm of scientists in the basic science arena. It is imperative that we focus on fundamental aspects of the genetics and biochemistry of AD; insights gained from these basic approaches will, indoubtedly, impact on our understanding of underlying pathogenetic mechanisms and the development and testing of new therapies for this devastating human illness.
ACKNOWLEDGEMENTS This work was supported by grants from the U.S. Public Health Service (NIH NS 20471 and AG 05146) and the Adler Foundation.
REFERENCES l. Anderson, J. P.; Esch, F. S.; Keim, P. S.; Sambamurti, K.; Lieberburg, I.; Robakis, N. K. Exact cleavage site of Alzheimer amyloid precursor in neuronal PC-12 cells. Neurosci. Len. 128:126-128; 1991. 2. Cai, X.-D.; Golde, T. E.; Younkin, S. G. Release of excess amyloid 13 protein from a mutant amyloid 13 protein precursor. Science 259: 514-516; 1993. 3. Chartier-Harlin, M.-C.; Crawford, F.; Houlden, H.; Warren, A.; Hughes, D.; Fidani, L.; Goate, A.; Rossor, M.; Roques, P.; Hardy, J.; Mullan, M. Early onset Alzheimer's disease caused by mutations at codon 717 of the 13-amyloid precursor protein gene. Nature 353: 844-846; 1991. 4. Citron, M.; Oltersdorf, T.; Haass, C.; McConlogue, L., Hung, A. Y.; Seubert, P.; Vigo-Pelfrey, C.; Lieberburg, I.; Selkoe, D. J. Mutation of the 13-amyloid precursor protein in familial Alzheimer's disease increases 13-protein production. Nature 360:672-674; 1992. 5. Cole, G. M.; Huynh, T. V.; Saitoh, T. Evidence for lysosomal processing of amyloid 13-protein precursor in cultured cells. Neurochem. Res. 14:933-939; 1989. 6. Esch, F. S.; Keim, P. S.; Beanie, E. C.; Blacher, R. W.; Culwell, A. R.; Oltersdorf, T.; McClure, D.; Ward, P. J. Cleavage of amyloid 13peptide during constitutive processing of its precursor. Science 248: 1122-1124; 1990. 7. Goate, A.; Chartier-Harlin, M.-C.; Mullah, M.; Brown, J.; Crawford, F.; Fidani, L.; Giuffra, L.; Haynes, A.; Irving, N.; James, L.; Mant, R.; Newton, P.; Rooke, K.; Roques, P.; Talbot, C.; Pericak-Vance, M.; Roses, A.; Williamson, R.; Rossor, M.; Owen, M.; Hardy, J. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature 349:704-706; 1991. 8. Golde, T. E.; Estus, S.; Younkin, L. H.; Selkoe, D. J.; Younkin, S. G. Processing of the amyloid protein precursor to potentially amyloidogenic derivatives. Science 255:728-730; 1992. 9. Haass, C.; Koo, E. H.; Mellon, A.; Hung, A. Y.; Selkoe, D. J. Targeting of cell-surface 13-amyloid precursor protein to lysosomes: AIten.tative processing into amyloid-bearing fragments. Nature 357:500503; 1992. 10. Haass, C.; Schlossmacher, M. G.; Hung, A. Y.; Vigo-Pelfrey, C.; Mellon, A.; Ostaszewski, B. L.; Lieberburg, I.; Koo, E. H.; Schenk, D.; Teplow, D. B.; Selkoe, D. J. Amyloid 13-peptide is produced by cultured cells during normal metabolism. Nature 359:322-325; 1992. 11. Hendricks, L.; van Duijn, C. M.; Cras, P.; Cruts, M.; Van Hul, W.; van Harskamp, F.; Warren, A.; Mclnnis, M. G.; Antonarakis, S. E.; Martin, J.-J.; Hofman, A.; Van Broeckhoven, C. Presenile dementia and cerebal haemorrhage linked to a mutation at codon 692 of the 13-amyloid precursor protein gene. Nature Genet. 1:218-221; 1992.
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